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    • To study film morphology in Fig we

    2018-10-26

    To study film morphology in Fig. 1 we show the optical microscopy image of M-L2 (a) and M-L3 (b) samples. From Fig. 1(a) it can be appreciated the uniform coverage of the support. Nevertheless, the surface is very rough, showing micrometric clusters and crystalline inclusions that can be recognized by the regular shape (square or rectangular). From this micrograph it can be inferred that the starting solution was too concentrated to be able to get a good deposition, with lipase dispersed at or almost at molecular level. In order to improve film morphology, in sample M-L3 the concentration of both solutes was lowered, maintaining constant their ratio. The optical micrograph of the sample M-L3 showed in Fig. 1(b) displays the absence of micrometric features. The lipase film is not optically visible, but its presence is evidenced from its AFM (Atomic Force Microscope) image presented in Fig. 2 in a 2μm×2μm window. The structural characteristics of the MAPLE deposited lipase were studied by means of FTIR spectroscopy. FTIR spectroscopy is widely used for studying the secondary structure of proteins [34–36]. Indeed different BQ-788 sodium salt bands arising from the protein backbone depends on the structural context (α-helices, β-sheets, turns) of the bond involved in the vibration. Among these, of particular utility is the band designated as Amide I between 1600 and 1700cm−1, which originates primarily from the stretching vibrations of the CO bond. This band is due to the overlapping of different modes depending of the nature of hydrogen bonding CO⋯HN, determined by the secondary structure of the protein. In details, three main secondary structure elements can be observed at 1650–1660cm−1 (α-helices), 1620–1650cm−1 (β-sheets) and 1660–1690cm−1 (turns and other disordered structures). At lower wavenumbers, 1610–1620cm−1, absorption is attributed to strongly intermolecular hydrogen-bonded β-sheets structures [37]. Quantitative analysis of the protein secondary structure was performed by assuming that the amide I band was the linear combination of Gaussian components arising from the various secondary structure elements. The number and initial positions of these components were set from the second derivative spectra, obtained after a binomial 5 point smoothing of the spectra (Savitzky–Golay method). Curve fitting was then performed using a fixed bandwidth (12cm−1) and a Gaussian profile. The intensity of each component was determined by the best fit results. The FTIR spectra of the three samples in the region 1525–1775cm−1, together with the amide I curve fitting into its Gaussian component, are shown in Fig. 3. As can be seen, the peak representing aggregated β-strands, i.e. stretching vibrations of intermolecular H-bonded CO in self-association at 1610–1620cm−1, lose progressively intensity by going from M-L1 to M-L3 in favor of the intensity of the peaks representing native secondary structure elements. Each component was correlated with the amount of peptide bonds in the structural unit from which the component originated, by dividing the area under each component by the total area [38]. Quantitative results of the curve fitting procedure for the three samples under study and Gaussian component attribution are reported in Table 2. Peaks before 1600cm−1 and after 1700cm−1 are attributed to the side-chains vibrations [38,34] and are not included in the calculation. In particular, the Gaussian peak appearing after the deconvolution at 1585cm−1 is due to COO− antisymmetric stretching of Asp and Glu carboxylate groups [38]. From the data in Table 2 it can be inferred that the sample deposited without the addition of m-DOPA (M-L1) showed a high degree of unfolding/aggregation/self-association (1624cm−1) of lipase molecules, especially at the expense of the secondary structure elements β-sheets and turns. In fact, in the native conformation of Candida Rugosa Lipase α-helices accounts for about 40%, while β-sheets for 23%, and turns for 28% as predicted by FTIR [40]. Adding m-DOPA reduced the percentage of self-association. Decreasing the concentration of the solute in the target (sample M-L3) further reduced the unfolding/aggregation occurring during the MAPLE process and the α-helices proportion almost reached that of the native conformation indicating that aggregation mainly occurred at the expenses of β-sheets. However some degree of unfolding/aggregation was still present in M-L3 sample, as indicated in Table 2.